Paralogous transcription factors, TT8a and TT8b, control soybean seed coat and hilum coloration.
Seed coat and hilum pigmentation represent commercially and nutritionally significant traits in soybean, determined by anthocyanin and proanthocyanidin accumulation. Despite their agricultural importance, the spatial regulatory mechanisms governing these flavonoid deposition patterns are not fully understood. In this study, we identified soybean TT8a/b as central bHLH transcription factors coordinating soybean seed coloration through integrative transcriptomic co-expression network analysis. TT8a/b exhibit specific spatiotemporal expression restricted to early seed developmental stages. CRISPR-generated soybean tt8a/b double mutants displayed complete hilum depigmentation and a significant reduction in anthocyanins, firmly establishing these transcription factors as master regulators of pigmentation in soybean seeds. Conversely, TT8a/b overexpression using their endogenous promoters significantly enhanced pigmentation intensity, demonstrating their dosage-dependent regulation. Transcriptomic analysis revealed near-complete suppression of key flavonoid biosynthetic genes in tt8a/b mutants, further validating direct regulatory influence. Population genetic analyses revealed that TT8a underwent strong selection during soybean domestication, and this selection gave rise to light-colored seed coats and hila. Our discovery of the decisive role of TT8a/b in seed coat and hilum pigmentation yields fundamental insights into plant flavonoid patterning and provides precision tools for breeding nutritionally enhanced and visually distinct soybean cultivars.